Medium for culturing human olfactory neurons

ABSTRACT

The present invention relates to a culture of human olfactory neurons. The neurons may display a normal neuronal pathology or a pathology characteristic of a generalized central nervous system disease. The cultured neurons can be used for neurotoxicity tests, screening for therapeutic drugs and anti-viral agents.

This is a continuation of application Ser. No. 07/487,894, filed Mar. 6,1990 now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to cultures of human olfactory neurons andto methods of screening agents for therapeutic and toxic effects usingsuch cultures.

2. Background Information

Neurons have eluded the attempts of researchers to maintain them incontinuous cultures largely because most neurons do not divide and/orproliferate. Investigators have been able to maintain fetal or newbornrat neurons in culture, however, the neurons are static, do not divideand die after several months.

Olfactory sensory neurons, which are of central origin and areconsidered part of the central nervous system, do divide Talamo et al.,Nature 337:736-739, (1989)!. In fact, olfactory neurons from rats havebeen cultured Coon et al., PNAS USA 86:1703-1707, (1989)!. However, ratsdo not develop human neurologic diseases, such as Alzheimer's diseaseand other human mental illnesses. Therefore, cultures of rat neurons cannot be used to test for potential therapeutic agents for these humandiseases. Furthermore, rats have different tolerance levels of toxiccompounds than humans. Therefore, the neurotoxicity of various agents tohumans could be more accurately determined using cultured human neuronsthan cultured rat neurons.

Cell cultures of cancer cells, such as human neuroblastoma cells, alsoexist. However, these cells tend to be undifferentiated and do notexhibit a strong neuronal phenotype. Neuroblastomas are not of centralnervous system origin. Furthermore, neuroblastomas have not beencultured from individuals with generalized central nervous systemdiseases (ie, neuro/psychiatric illnesses).

Human olfactory neurons, being primarily, of central nervous systemorigin from healthy individuals and individual with neuro/psychiatricillnesses would offer better systems for testing agents for therapeuticand toxic effects.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide humanolfactory neuron cultures from individuals without central nervoussystem diseases and from individuals with diseases of the centralnervous system.

It is another object of the present invention to provide a method forassessing the potential neurotoxicity and potential therapeutic effectsof agents in humans without employing live animals.

It is a further object of the present invention to provide a method forstudying generalized diseases of the central nervous system.

Various other object and advantages of the present invention will beapparent from the drawings and the following description of theinvention.

In one embodiment, the present invention relates to a culture ofreplicating mammalian olfactory neurons, such as human. The neurons maydisplay normal neuronal pathology or they may display pathologycharacteristic of a central nervous system disease.

In another embodiment, the present invention relates to a method ofreplicating mammalian olfactory neurons comprising contacting mammalianneurons with a growth medium and maintaining the neurons and mediumunder conditions allowing replication.

In a further embodiment, the present invention relates to a method ofscreening drugs for their ability to reverse or eliminate expression ofcentral nervous system disease pathology comprising contacting culturedhuman neurons which show pathological characteristics of a centralnervous system disease with a drug under conditions such that reversalor elimination of the disease pathology can be effected.

In yet another embodiment, the present invention relates to a method ofscreening drugs for their ability to inhibit virus activity on neuronscomprising contacting cultured human neurons with a drug underconditions such that inhibition of viral function can be effected.

In yet a further embodiment, the present invention relates to a methodof testing for neurotoxicity of an agent comprising contactingreplicating human olfactory neurons with an agent and determining thetoxic effect of the agent on the neurons.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the morphology of neurons grown in culture underconditions allowing for growth in three dimensions.

1B shows the same neurons when grown on a basement membrane coated flatsurface.

1C shows epithelial cells grown from the tissue sample on a basementmembrane coated flat surface.

1D and 1E show other cells not fully characterized grown on a basementmembrane coated flat surface.

FIG. 2 shows Western Blots of neuron specific proteins.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

The present invention relates to cultures of human olfactory neurons anduses thereof. Cultures of human olfactory neurons are established fromhuman tissue samples containing neurons, such as nasal epithelium.Suitable tissue samples are obtained from the cadavers of healthyindividuals that is to say individuals without any central nervoussystem diseases. Cultured neurons thus obtained display normal neuronalpathology and express several neuron specific proteins (eg. neuronspecific enolase, neurofilament (68 KD form) and tau protein).

In another embodiment of the present invention, cultures of humanolfactory neurons are established from a tissue sample obtained from thecadavers of individuals with Alzheimer's disease. It is anticipated thatthese cultures of neurons will display the pathology associated withAlzheimer's disease Talamo et al., Nature 337:736-739, (1989)!. As oneskilled in the art will appreciate, cultures of neurons can beestablished using the method of the present invention from an individualafflicted with any generalized central nervous system disease whichincludes neurological and psychiatric illnesses such as Parkinson'sdisease, Tay-Sachs disease, schizophrenia, manic-depression, depressionor mental retardation. A generalized central nervous system diseaseaffects many types of neurons in the central nervous system includingolfactory neurons. These cultured neurons may display the pathology ofthe disease affecting the individual from which the culture was grown.

In a preferred embodiment, the neuron cultures are established utilizinga basement membrane. The basement membrane consists mainly of lamininand collagen. Therefore, the present invention contemplates culturedhuman neurons which are initially grown and/or maintained on a membraneof laminin, collagen, related peptides thereof or a combination of theforegoing. When maintained under conditions allowing growth in threedimensions in the membrane the neurons appear more differentiated.

The present invention also relates to cultures of transformed humanolfactory neurons. One skilled in the art can easily transform andthereby immortalize cultured human olfactory neurons of the presentinvention without undue experimentation. Cultured neurons of the presentinvention can be transformed using, for example, the basic protocoldescribed by Fredericksen et al. Neuron 1:439-448, (1988)!. Neurons canbe transformed using a retroviral vector, such as pZipNeoSVC(X), whichcontains a temperature sensitive form of the SV40 large T antigen tsA58; the large T antigen is an oncogene. The transformed neurons willproliferate indefinitely in a relatively undifferentiated state at thepermissive temperature of 33° C. but will be able to differentiate intoneurons at the nonpermissive temperature of 39° C.

In another embodiment of the present invention, cultured neurons from anindividual with a central nervous system disease are used for screeningdrugs for their ability to reverse or eliminate the disease pathology.The cultured neurons are contacted with various drugs under conditionssuch that reversal or elimination of the disease pathology can beeffected by the drug if it has the ability to do so.

For example, levels of dopamine receptors and/or receptor function incells from a schizophrenic individual can be compared to that of normalindividuals. If differences are found, then these differences can beused in assays to study the disease and to screen for drugs that correctthe alteration. The presence of paired helical filaments or otherAlzheimer's related antigens such as A68 can be used to assay forAlzheimer's disease pathology Wolozin et al., Science 232:648-650,(1986)!. In manic-depression alterations in G protein function arehypothesized. Neurons of the present invention cultured from anindividual with manic-depression can be used to screen for drugscorrecting the G protein misfunction. Cultured neurons can also manifestthe disorder present in gangliosidoses like Tay-Sachs disease, inheritedforms of mental retardation or other inherited neurologic diseases Adamset al., Principles of Neurology. (McGraw-Hill, New York. 1985).pp.718-759!. Neurons of the present invention can be used to screen fordrugs decreasing the build up of gangliosides.

Neurons of the present invention which naturally or through amodification secrete dopamine can be used for injection into thesybstantia nigra of individual with Parkinson's disease.

In another embodiment of the present invention, cultured neurons areused to screen for drugs that inhibit viral activity in neurons. Neuronsof the present invention are contacted with a virus which naturallyinfects human neurons, such as the herpes virus or cytomegalovirus(CMV), and with various drugs under conditions such that inhibition ofviral function can be effected. Drugs which inhibit virus attachment,penetration, replication or toxicity can be screened for and could beuse in treatment or prevention of such infections.

Rat olfactory neurons can be stimulated to release the calcitonin generelated peptide (CGRP) by altering potassium levels when incubated witha neurotoxin. Release of CGRP may be a sensitive indicator ofneurotoxicity. Human olfactory neurons of the present inventioncontaining CGRP or any other neurotransmitter can be used to assess theneurotoxicity of an agent by detecting alterations of basal andpotassium evoked neurotransmitter release into the medium in thepresence and absence of the agent. Neurotoxicity may correlate with adecline in neurotransmitter release over time as neuronal functiondeteriorates.

In another embodiment of the present invention, the cultured neurons areused for testing the neurotoxicity of an agent in humans. As one skilledin the art will appreciate, determining the effect of the agent on anyof a number of neuronal characteristics will reflect that neurotoxicityof the agent. For example, the concentration of the agent producingdeath in 50% of the neurons (LD50) can be determined using methods knownin the art. The effect of the agent on neuronal growth characteristicssuch as population doubling time and length of processes also reflectthe toxicity of the agent to humans.

For purposes of illustrating a preferred embodiment of the presentinvention, in the following non-limiting example, cultures of olfactoryneurons from individual without central nervous system diseases and fromindividuals with Alzheimer's disease were established. It is, however,to be understood that the discussion generally applies to establishedcultures of neurons from individuals with any central nervous systemdisease.

EXAMPLE

Procurement of Olfactory Epithelium

Olfactory epithelium was procured from cadavers of healthy individualsand Alzheimer's individuals using a cup shaped curette 6 cm in lengthand with a curette size of 5 or 7.5 mm (depending on the size of thenasal passage).

One of the two approaches (Anterior or Posterior) was taken to obtainepithelium containing olfactory neurons. In the "Anterior" approach, thecurette was advanced into the nasal vestibule. The curette was thenplaced adjacent to the first nasal turbinate (concha) encountered. Thefirst turbinate was usually encountered after the curette was advancedabout 3 or 4 cm into the nasal vestibule.

The turbinate was scraped 3 or 4 times with the curette. Olfactoryepithelium, yellower than the surrounding tissue was obtained. Since theolfactory tissue lies over the bone, the presence of some boneassociated with the scrapings is a good sign.

The tissue was placed in modified L-15 transport medium containingpolyvinylpyrrolidone-360 200 mg/l, glutathione 0.79 mg/l,2-mercaptoethanol 50 mg/l, fetal bovine serum 1%, penicillin 200 U/ml,streptomycin sulfate 200 mcg/ml (all above agents were from Sigma orGIBCO) and fungizone 2.5 ug/ml (Squibb). The tissue was transported onice. However, the sample was not frozen since freezing kills the tissue.

As an alternative to the modified L-15 transport medium described above,the L-15 transport medium may be modified to contain some or all of theagents described by Kischer et al. Cytotechnology 2:181-185, (1989)!.

Procurement of olfactory epithelium using the "Posterior" approach wassomewhat more difficult than with the "Anterior" approach.

The olfactory epithelium lies underneath the cribriform plate and sendsaxons up through the plate into the olfactory bulb. Therefore, theportion of the cribiform plate immediately underneath the olfactory bulbwas removed and the tissue underneath collected. It was not necessary toseparate the tissue from the attached bone; however, the dura had to beremoved.

The collected tissue was transported as described above in the"Anterior" procurement approach.

Olfactory Neuron Culturing

The olfactory neurons were grown using the basic method described byCoon et al. PNAS USA 86:1703-1707, (1989); see also Ambesi-Impiombato etal. PNAS USA 77:3455-3459, (1980)!. The collected tissue was cut into 1mm×1 mm pieces and put under a reconstituted basement membranepreparation available as "matrigel" (Collaborative Research Inc.) andkept in Coon's 4506 medium.

Coon's 4506 medium uses modified Ham's F-12 medium as a base. Coon's4506 is made by first preparing Coon's modified Ham's F-12 to includethe amino acids, salts and minerals described below in the Table 1. Themodified Ham's F-12 is then further modified by including the itemslisted below in Table 2 to generate Coon's 4506 medium.

                  TABLE 1    ______________________________________    Coon's Modified Ham's F-12    neuroblast formulation**    Component           grams per liter    ______________________________________    L-Alanine           0.018    L-Arginine HCl      0.420    L-Asparagiane Anhyd 0.030    L-Aspartic Acid     0.026    L-Cysteine HCl--H.sub.2 O                        0.070    L-Glutamic Acid     0.030    L-Glutamine         0.290    Glycine             0.160    L-Histidine HCl--H.sub.2 O                        0.042    L-Isoleucine        0.008    L-Leucine           0.026    L-Lysine HCl        0.073    L-Methionine        0.009    L-Phenylalanine     0.010    L-Proline           0.070    L-Serine            0.021    L-Threonine         0.024    L-Tryptophan        0.004    L-Tyronsine 2Na     0.016    L-Valine            0.023    D-Glucose           2.00    Biotin              0.00007    D-Ca Pantothenate   0.0005    Choline Chloride    0.014    Folic Acid          0.001    Myo-inositol        0.036    Niacinamide         0.00004    Pyridoxine HCl      0.00006    Riboflavin          0.00004    Thiamine HCl        0.00029    Vitamin B-12        0.0014    Putrescine 2HCl     0.0003    Na Pyruvate         0.220    Na Hypoxanthine     0.0047    Thymidine           0.0007    L-Ascorbic Acid     0.045    Linoleic Acid       0.00009    Lipoic Acid         0.0002    Phenol Red          0.0012    Sodium Chloride     7.530.    Potassium Chloride  0.230**    Sodium Phosphate Dibasic Anhyd                        0.135    or as - 7H.sub.2 O  0.250    Potassium Phosphate Monobasic                        0.068    Magnesium Chloride - 6H.sub.2 O                        0.081**    Magnesium Chloride - 7H.sub.2 O                        0.025**    Calcium chloride    0.0**    Cupric Sulfate - 6H.sub.2 O                        0.000002    Ferrous Sulfate - 7H.sub.2 O                        0.0008    Zinc Sulfate - 7H.sub.2 O                        0.000144    ______________________________________

                  TABLE 2    ______________________________________    Coon's 4506 Medium    ______________________________________    MODIFIED F12 (mFl2. neuroblast formulation)     Ham's F12 with the following changes:!    2 times the concentration of AMINO ACIDS,    PYRUVATE    ASCORBATE             45 μg/ml    HANK'S SALTS in place of those specified by    Ham    Hank's formulation further modified so as to    contain    Mg++-0.48mM    NO ADDED Ca++(Final ≦ 0.1 mM from    extracts)    KCl reduced           0.230 mg/ml    FOLIC ACID            0.001 mg/ml    HYPOXANTHINE          0.0047 mg/ml    THYMIDINE             0.0007 mg/ml    GLUCOSE               2 mg/ml    GALACTOSE             0.5 mg/ml    NO LINOLEIC ACID    ADDITIVES to make 4506 from mF12 base:    FETAL BOVINE SERUM (GIBCO)                          6.0%    TRANSFERRIN (HUMAN)   5 μg/ml    INSULIN (Na+)         1 μg/ml    HYDROCORTISONE        3.5 μg/ml    SELENOUS ACID         2.5 μg/ml    THYROXINE T.sub.3     40 μg/ml    GENTAMYCIN SO.sub.4 (GIBCO)                          50 μg/ml    EXTRACTS (final concentrations):    BOVINE HYPOTHALAMUS   150 μg protein/ml    BOVINE pituitaryMUS   50 μg protein/ml    CULTURES made in droplets or on plates coated with:    "BASEMENT MEMBRANE"   -150 μg protein/ml    (e.g. "Martrigel" Collaborative Research, Inc.)    ______________________________________

After several weeks of culture, neurons began to grow. A variable numberof tissue pieces, between 10-100% grew out neurons. Neuronal cultureswere selected based on the morphology of the cells. Other types of cellsthat grew out were epithelial, glandular and a spindly type. (See FIG.1). The basement membrane functioned to inhibit growth of other celltypes and promote neuronal growth.

The neurons were collected as in Coon et al. PNAS USA 86:1703-1707,(1989)! and grown in cell culture dishes coated with basement membrane.Dishes were coated with basement membrane by spreading cold basementmembrane on the dish and then leaving the dish at 37° C. for at least10-20 minutes. The Coon's 4506 medium was changed twice a week. Cellswere not allowed to remain confluent for more than 2 days. The neuronswere harvested from the dishes by treating the neuron cultures with aprotease solution, Dispase (Boehringer-Mannheim, Indianapolis) for 1 hrat 37° C. The medium containing the detached cells was spun down at 1000rpm for 10 min, the supernatant removed and then the cells wereresuspended in appropriate medium. Cells were always placed onto platescoated with basement membrane solution.

For storage, cells were in Coon's 4506 medium containing 10%dimethylsulfoxide. Cells were frozen down under liquid nitrogen.

Clonal colonies of neurons were also obtained by diluting harvestedneurons in Coon's 4506, growing them on basement membrane coated dishes,isolating individual colonies using cloning cylinders (BellCo) and thenharvesting individual colonies as described above.

Coon's 4506 medium was required for initial growth of the neurons. Onceestablished, the culture may be able to be maintained using othermediums such as Keratinocyte Growth Medium (Clonetics, San Diego)instead of the Coon's 4506.

Identification of Neuronal Cells

Neuronal cells were identified using immunocytochemistry and westernblot methodologies well known in the art. The cells were shown toexpress several neuron-specific proteins: Neuron Specific Enolase,Neurofilament (68 KD form) and Tau Protein (see FIG. 2).

From a tissue sample containing only neurons, a pure neuron populationwas grown out without the aid of the basement membrane. The Coon's 4506had an increased amount of fetal bovine serum (20%) and culture growthwas slower.

Human olfactory neurons strains 1402 and 90-1 were deposited on Mar. 6,1990 under the Budapest Treaty at the American Type Culture Collection,12301 Parklawn Drive, Rockville, Md. 20852.

The entire contents of all references cited hereinabove are herebyincorporated by reference.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention.

What is claimed is:
 1. A tissue culture medium designated Coon's 4506.